The present invention relates generally to manipulation of genetic materials and, more particularly, to methods and materials useful in subjecting the transcription of particular DNA sequences to selective regulation by external control.
"Genetic materials" may be broadly defined as those substances which program for and guide the manufacture of cellular (and viral) constituents and the responses of cells and viral particles to environmental changes. The genetic material of all living cells and viruses (except the so-called "RNA viruses") comprises a long chain, polymeric substance known as deoxyribonucleic acid ("DNA"). The repeating units of the DNA polymer are known as nucleotides. Each nucleotide consists of one of four nucleic acids (adenine, guanine, cytosine and thymine) bound to a sugar (deoxyribose) which has a phosphate group attached. Ribonucleic acid ("RNA") is a polymeric nucleotide comprising the nucleic acids, adenine, guanine, cytosine and uricil, bound to a ribose molecule having an attached phosphate group.
Most simply put, the programming function of genetic materials is generally effected through a process whereby DNA nucleotide sequences (genes) are "transcribed" into relatively unstable messenger RNA ("mRNA") polymers which, in turn, serve as templates for formation of structural, regulatory and catalytic proteins from amino acids. Protein synthesis is thus the ultimate form of "expression" of the programmed genetic message provided by the DNA sequence of a gene.
Certain DNA sequences which usually "precede" a gene in a DNA polymer provide a site for initiation of the transcription into mRNA. These are referred to as "promoter" sequences. Other DNA sequences, also usually "upstream" of a gene in a given DNA polymer, bind proteins that determine the frequency (or rate) of transcription initiation. These other sequences are referred to as "regulator" sequences. Thus, sequences which precede a selected gene (or series of genes) in a functional DNA polymer and which operate to determine whether the transcription (and eventual expression) of a gene will take place are collectively referred to as "promoter/regulator" DNA sequences.
The promoter/regulator sequences of genes are clearly susceptible to enormous structural and functional variation and, in fact, only a few such sequences in rather simple genetic systems have been thoroughly structurally and operationally characterized. Promoter/regulator sequences, in general, serve to regulate gene transcription in response to chemical (and sometimes, physical) environmental conditions in and around the cell. Many generalized "models" for the action of promoter/regulator operation in gene transcription and eventual expression in simple, prokaryotic systems have been proposed. One such model posits a "repressor" gene and a regulator sequence or "operator" sequence near the promoter of another gene. According to this model, transcription of the repressor sequence results in expression of a repressor protein which selectively binds to the operator sequence to effectively preclude gene transcription of the selected gene. An environmental "signal" (e.g., increased concentration of a chemical acted upon by the protein product of the gene in question) may operatively inactivate the repressor protein, blocking its ability to bind to the operator sequence in a way which would interrupt transcription of the gene. Increased concentrations of a substrate could be seen as operating to "induce" synthesis of the protein which catalyzes its breakdown.
Another generalized model of operation of promoter/regulator sequences in the regulation of gene transcription posits formation of an initially inactive form of repressor protein by the repressor DNA sequence. Such an inactive form could not bind to an operator DNA sequence (and disrupt selected gene transcription) until it is combined with some other substance present in the cell. The other substance could be, for example, a compound which is the product of a reaction catalyzed by the protein coded for by the selected gene. Increased concentrations of such a reaction product in the cell would thus operate to repress the potential overproduction of proteins responsible for the product's synthesis. In these examples, the regulator protein functions to inhibit transcription. Other regulatory proteins have been described which potentiate or activate transcription of specific DNA sequences. Thus, there are examples of both negative and positive control proteins and corresponding regulatory DNA sequences.
Similar "models" for the operation of promoter/regulator DNA sequences in eukaryotic cells have been proposed. See, e.g., Brown, "Gene Expression in Eukaryotes", Science, 211, pp. 667-674 (1981).
Among the basic problems of genetic engineering is the isolation and preparation of multiple copies of selected gene sequences of interest, together with the promoter/regulator DNA sequences which normally affect their transcription in the cells from which they are isolated. Another basic problem of genetic engineering is the insertion and stable incorporation of DNA sequences into cells in a manner which will permit external regulation of the transcription of the gene sequences and their expression.
Significant advances in the isolation and copying of selected DNA sequences have been made possible by the use of restriction endonuclease enzymes (which are capable of effecting site specific cuts in DNA polymers) and ligating enzymes (which serve to fuse DNA sequences together). DNA sequences of interest are usually incorporated into "vectors" of plasmid or viral origin that allow selective replication in a suitable host cell (for example, bacteria, yeast, or mammalian cells). When these vectors with DNA sequences of interest are introduced into cells of higher animals or plants, they may either be maintained as extrachromosomal elements or incorporated into the chromosomes.
Most genetic engineering activity to date has been directed toward the stable incorporation of exogenous DNA in prokaryotic cells such as bacteria and in the simpler eukaryotes such as yeasts, molds and algae. The hoped-for result of these experiments has been to provide not only a source of multiple copies of selected genes, but the large scale transcription and expression of commercially significant gene in the form of proteinacious products. See, e.g., Cohen, et al., U.S. Pat. No. 4,237,224; Manis, U.S. Pat. No. 4,273,875; and Cohen, U.S. Pat. No. 4,293,652. Work involving eukaryotic cells of higher organisms such as plants and animals has generally involved cells which are capable of continuous growth in culture.
Of significant interest to the background of the invention are numerous publications of prior investigations by the co-inventors and their co-workers relating to: (1) regulation of mammalian gene expression; and (2) introduction of purified genes into eukaryotic cells.
Specifically incorporated by reference herein for purposes of indicating the background of the invention and illustrating the state of the prior art are the following publications of co-inventor Palmiter and his co-workers: Durnam, et al., "Isolation and Characterization of the Mouse Metallothionein-I Gene", P.N.A.S., 77, pp. 6511-6515 (1980); Durnam, et al., "Transcriptional Regulation of the Mouse Metallothionein-I Gene by Heavy Metals", J. Biol. Chem., 256, pp. 5712-5716 (1981); Mayo, et al., "Gluocorticoid Regulation of Metallothionein-I mRNA Synthesis in Cultured Mouse Cells", J. Biol. Chem., 256, pp. 2621-2624 (1981); Hager, et al., "Transcriptional Regulation of Mouse Liver Metallothionein-I Gene by Glucocorticoids", Nature, 291, pp. 340-342 (1981); Glanville, et al., "Structure of Mouse Metallothionein-I Gene and Its mRNA", Nature, 292, pp. 267-269 (1981); and Beach, et al., "Amplification of the Metallothionein-I Gene in Cadmium Resistant Mouse Cells", P.N.A.S., 78, pp. 2210-2214 (1981). The foregoing all deal with the DNA sequence specifying production of a low molecular weight, metal-binding protein found in one or more forms in most vertebrate tissues. More particularly, the publications treat mouse metallothionein genes as well as their promoter/regulator DNA sequences and the responsiveness of the promoter/regulator sequences to metals and steroid hormones.
Additional publications of co-inventor Palmiter and his co-worker which are incorporated by reference herein are: McKnight, et al., "Transferrin Gene Expression, Regulation of mRNA Transcription in Chick Liver by Steroid Hormones and Iron Deficiency", J. Biol. Chem., 255, pp. 148-153 (1980); and Palmiter, et al., "Steroid Hormone Regulation of Ovalbumin and Conalbumin Gene Transcription, A Model Based Upon Multiple Regulatory Sites and Intermediary Proteins", J. Biol. Chem., 256, pp. 7910-7916 (1981).
Also incorporated by reference herein is a publication of co-inventor Brinster and his co-workers dealing with microinjection of plasmids into germinal vesicles of mouse oocytes or pronuclei of fertilized mouse ova, Brinster, et al., "Mouse Oocytes Transcribe Injected Xenopus 5S RNA Gene", Science, 211, pp. 396-398 (1981).
Also pertinent to the background of the present invention and incorporated by reference herein, are the publications of Illmensee, et al., Cell, 23, pp. 9-18 (1981) and Gordon, et al., P.N.A.S., 77, pp. 7380-7384 (1981) which respectively treat injection of nuclei into enucleated mouse eggs and introduction of plasmids containing the herpes thymidine kinase gene and SV40 (Simian virus) sequences into mice. Finally, the recent publication of Wagner, et al. appearing in P.N.A.S., 78 pp. 5016-5020 (1981) and treating incorporation of the human .beta.-globin gene and a functional viral thymidine kinase gene into developing mice, is pertinent to the background of the present invention.